Methods for the synthesis of arrays of DNA probes

ABSTRACT

The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing reflective projection optics. The projection optics project a light image onto the active surface of the substrate to deprotect linker molecules thereon. A first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different light image, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior application Ser. No.09/637,891 filed Aug. 9, 2000, which is a continuation of priorapplication Ser. No. 09/253,460 filed Feb. 22, 1999, which applicationclaimed the benefit of provisional patent application Ser. No.60/075,641, filed Feb. 23, 1998.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with United States government support awarded bythe following agencies: DOE Grant Nos.: DE-FG07-96ER13938; P0062242-02;DE-FG02-96ER45569; 63040304; P071760302; NSF Grant Nos.: IBN-9706552;ECS-9317745; INT-960289; ONR DOD-Navy Grant# N00014-97-1-0460; DOD-ArmyGrant # DAAH04-95-1-0456; USDA AGRICCREE Grant No.: 95-37304-2364. TheUnited States has certain rights in this invention.

FIELD OF THE INVENTION

This invention pertains generally to the field of biology andparticularly to techniques and apparatus for the analysis and sequencingof DNA and related polymers.

BACKGROUND OF THE INVENTION

The sequencing of deoxyribonucleic acid (DNA) is a fundamental tool ofmodern biology and is conventionally carried out in various ways,commonly by processes which separate DNA segments by electrophoresis.See, e.g., Current Protocols In Molecular Biology, Vol. 1, Chapter 7,“DNA Sequencing,” 1995. The sequencing of several important genomes hasalready been completed (e.g., yeast, E. coli), and work is proceeding onthe sequencing of other genomes of medical and agricultural importance(e.g., human, C. elegans, Arabidopsis). In the medical context, it willbe necessary to “re-sequence” the genome of large numbers of humanindividuals to determine which genotypes are associated with whichdiseases. Such sequencing techniques can be used to determine whichgenes are active and which inactive either in specific tissues, such ascancers, or more generally in individuals exhibiting geneticallyinfluenced diseases. The results of such investigations can allowidentification of the proteins that are good targets for new drugs oridentification of appropriate genetic alterations that may be effectivein genetic therapy. Other applications lie in fields such as soilecology or pathology where it would be desirable to be able to isolateDNA from any soil or tissue sample and use probes from ribosomal DNAsequences from all known microbes to identify the microbes present inthe sample.

The conventional sequencing of DNA using electrophoresis is typicallylaborious and time consuming. Various alternatives to conventional DNAsequencing have been proposed. One such alternative approach, utilizingan array of oligonucleotide probes synthesized by photolithographictechniques is described in Pease, et al., “Light-GeneratedOligonucleotide Arrays for Rapid DNA Sequence Analysis,” Proc. Natl.Acad. Sci. USA, Vol. 91, pp. 5022-5026, May 1994. In this approach, thesurface of a solid support modified with photolabile protecting groupsis illuminated through a photolithographic mask, yielding reactivehydroxyl groups in the illuminated regions. A 3′ activateddeoxynucleoside, protected at the 5′ hydroxyl with a photolabile group,is then provided to the surface such that coupling occurs at sites thathad been exposed to light. Following capping, and oxidation, thesubstrate is rinsed and the surface is illuminated through a second maskto expose additional hydroxyl groups for coupling. A second 5′ protectedactivated deoxynucleoside base is presented to the surface. Theselective photodeprotection and coupling cycles are repeated to build uplevels of bases until the desired set of probes is obtained. It may bepossible to generate high density miniaturized arrays of oligonucleotideprobes using such photolithographic techniques wherein the sequence ofthe oligonucleotide probe at each site in the array is known. Theseprobes can then be used to search for complementary sequences on atarget strand of DNA, with detection of the target that has hybridizedto particular probes accomplished by the use of fluorescent markerscoupled to the targets and inspection by an appropriate fluorescencescanning microscope. A variation of this process using polymericsemiconductor photoresists, which are selectively patterned byphotolithographic techniques, rather than using photolabile 5′protecting groups, is described in McGall, et al., “Light-DirectedSynthesis of High-Density Oligonucleotide Arrays Using SemiconductorPhotoresists,” Proc. Natl. Acad. Sci. USA, Vol. 93, pp. 13555-13560,November 1996, and G. H. McGall, et al., “The Efficiency ofLight-Directed Synthesis of DNA Arrays on Glass Substrates,” Journal ofthe American Chemical Society. 119, No. 22, 1997, pp. 5081-5090.

A disadvantage of both of these approaches is that four differentlithographic masks are needed for each monomeric base, and the totalnumber of different masks required are thus four times the length of theDNA probe sequences to be synthesized. The high cost of producing themany precision photolithographic masks that are required, and themultiple processing steps required for repositioning of the masks forevery exposure, contribute to relatively high costs and lengthyprocessing times.

SUMMARY OF THE INVENTION

In accordance with the present invention, the synthesis of arrays of DNAprobe sequences, polypeptides, and the like is carried out rapidly andefficiently using patterning processes. The process may be automated andcomputer controlled to allow the fabrication of a one or two-dimensionalarray of probes containing probe sequences customized to a particularinvestigation. No lithographic masks are required, thus eliminating thesignificant costs and time delays associated with the production oflithographic masks and avoiding time-consuming manipulation andalignment of multiple masks during the fabrication process of the probearrays.

In the present invention, a substrate with an active surface to whichDNA synthesis linkers have been applied is used to support the probesthat are to be fabricated. To activate the active surface of thesubstrate to provide the first level of bases, a high precisiontwo-dimensional light image is projected onto the substrate,illuminating those pixels in the array on the substrate active surfacewhich are to be activated to bind a first base. The light incident onthe pixels in the array to which light is applied deprotects OH groupsand makes them available for binding to bases. After this developmentstep, a fluid containing the appropriate base is provided to the activesurface of the substrate and the selected base binds to the exposedsites. The process is then repeated to bind another base to a differentset of pixel locations, until all of the elements of the two-dimensionalarray on the substrate surface have an appropriate base bound thereto.The bases bound on the substrate are protected, either with a chemicalcapable of binding to the bases or with a layer(s) of photoresistcovering all of the bound bases, and a new array pattern is thenprojected and imaged onto the substrate to activate the protectingmaterial in those pixels to which the first new base is to be added.These pixels are then exposed and a solution containing the selectedbase is applied to the array so that the base binds at the exposed pixellocations. This process is then repeated for all of the other pixellocations in the second level of bases. The process as described maythen be repeated for each desired level of bases until the entireselected two-dimensional array of probe sequences has been completed.

The image is projected onto the substrate utilizing an image formerhaving an appropriate light source that provides light to a micromirrordevice comprising a two-dimensional array of electronically addressablemicromirrors, each of which can be selectively tilted between one of atleast two separate positions. In one of the positions of eachmicromirror, the light from the source incident on the micromirror isdeflected off an optical axis and away from the substrate, and in asecond of the at least two positions of each micromirror, the light isreflected along the optical axis and toward the substrate. Projectionoptics receive the light reflected from the micromirrors and preciselyimage the micromirrors onto the active surface of the substrate.Collimating optics may be used to collimate the light from the sourceinto a beam provided directly to the micromirror array or to a beamsplitter, wherein the beam splitter reflects a portion of the beam tothe micromirror array and transmits reflected light from the micromirrorarray through the beam splitter. The light directly reflected from themicromirrors or transmitted through the beam splitter is directed toprojection optics lenses which image the micromirror array onto theactive surface of the substrate. Because the selectively addressablemicromirrors in the micromirror array may either fully reflect or fullydeflect the light provided to them, the image of the micromirror arrayexhibits a very high contrast between the “on” and “off” pixels. Themicromirrors may also be capable of being indexed to more than twopositions, in which case additional optics may be provided to allowexposure of more than one substrate using a single micromirror arraydevice. In addition, the micromirrors are capable of reflecting light atany wavelength without damage to them, allowing short wavelength light,including light in the range of ultraviolet to near ultraviolet light,to be utilized from the light source.

The micromirror array is operated under control of a computer whichprovides appropriate pixel address signals to the micromirror array tocause the appropriate micromirrors to be in their “reflect” or “deflect”positions. The appropriate micromirror array pattern for each activationstep in each level of bases to be added to the probes is programmed intothe computer controller. The computer controller thus controls thesequencing of the images presented by the micromirror array incoordination with the reagents provided to the substrate.

In one embodiment, the substrate may be transparent, allowing the imageof the micromirror array to be projected through the surface of thesubstrate that is opposite to the active surface. The substrate may bemounted within a flow cell, with an enclosure sealing off the activesurface of the array, allowing the appropriate reagents to be flowedthrough the flow cell and over the active surface of the array in theappropriate sequence to build up the probes in the array.

Further objects, features and advantages of the invention will beapparent from the following detailed description when taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a schematic view of an array synthesizer apparatus inaccordance with the present invention.

FIG. 2 is a schematic view of another array synthesizer apparatus inaccordance with the present invention.

FIG. 3 is a more detailed schematic view of a general telecentric arraysynthesizer apparatus in accordance with the invention.

FIG. 4 is an illustrative ray diagram for the refractive optics of theapparatus of FIG. 3.

FIG. 5 is a schematic view of a further embodiment of an arraysynthesizer apparatus in accordance with the invention in whichtelecentric reflective optics are utilized.

FIG. 6 is an illustrative ray diagram for the reflective optics of theapparatus of FIG. 5.

FIG. 7 is a top plan view of a reaction chamber flow cell which may beutilized in the array synthesizer apparatus of the invention.

FIG. 8 is a cross-sectional view through the reaction chamber flow cellof FIG. 7 taken generally along the lines 8-8 of FIG. 7.

FIG. 9 is an illustrative view showing the coating of a substrate with aphotolabile linker molecule.

FIG. 10 is an illustrative view showing the photo-deprotection of thelinker molecule and the production of free OH groups.

FIG. 11 is an illustrative view showing the coupling of markers to freeOH groups produced by the photo-deprotection of the linker molecules.

FIG. 12 is an illustrative view showing the coupling of DMT-nucleotideto free OH groups produced from photo-deprotection of the linkermolecules.

FIG. 13 is an illustrative view showing acid deprotection ofDMT-nucleotides.

FIG. 14 is an illustrative view showing the hybridization of poly-Aprobe labeled with fluorescein with poly-T oligonucleotide synthesizedfrom DMT-nucleotide-CEPs.

DETAILED DESCRIPTION OF THE INVENTION

With reference to the drawings, an exemplary apparatus that may be usedfor DNA probe array synthesis, polypeptide synthesis, and the like isshown generally at 10 in FIG. 1 and includes a two-dimensional arrayimage former 11 and a substrate 12 onto which the array image isprojected by the image former 11. For the configuration shown in FIG. 1,the substrate has an exposed entrance surface 14 and an opposite activesurface 15 on which a two-dimensional array of nucleotide sequenceprobes 16 are to be fabricated. For purposes of illustration, thesubstrate 12 is shown in the figure with a flow cell enclosure 18mounted to the substrate 12 enclosing a volume 19 into which reagentscan be provided through an input port 20 and an output port 21. However,the substrate 12 may be utilized in the present system with the activesurface 15 of the substrate facing the image former 11 and enclosedwithin a reaction chamber flow cell with a transparent window to allowlight to be projected onto the active surface. The invention may alsouse an opaque or porous substrate. The reagents may be provided to theports 20 and 21 from a conventional base synthesizer (not shown in FIG.1).

The image former 11 includes a light source 25 (e.g., an ultraviolet ornear ultraviolet source such as a mercury arc lamp), an optional filter26 to receive the output beam 27 from the source 25 and selectively passonly the desired wavelengths (e.g., the 365 nm Hg line), and a condenserlens 28 for forming a collimated beam 30. Other devices for filtering ormonochromating the source light, e.g., diffraction gratings, dichroicmirrors, and prisms, may also be used rather than a transmission filter,and are generically referred to as “filters” herein. The beam 30 isprojected onto a beam splitter 32 which reflects a portion of the beam30 into a beam 33 which is projected onto a two-dimensional micromirrorarray device 35. The micromirror array device 35 has a two-dimensionalarray of individual micromirrors 36 which are each responsive to controlsignals supplied to the array device 35 to tilt in one of at least twodirections. Control signals are provided from a computer controller 38on control lines 39 to the micromirror array device 35. The micromirrors36 are constructed so that in a first position of the mirrors theportion of the incoming beam of light 33 that strikes an individualmicromirror 36 is deflected in a direction oblique to the incoming beam33, as indicated by the arrows 40. In a second position of the mirrors36, the light from the beam 33 striking such mirrors in such secondposition is reflected back parallel to the beam 33, as indicated by thearrows 41. The light reflected from each of the mirrors 36 constitutesan individual beam 41. The multiple beams 41 are incident upon the beamsplitter 32 and pass through the beam splitter with reduced intensityand are then incident upon projection optics 44 comprised of, e.g.,lenses 45 and 46 and an adjustable iris 47. The projection optics 44serve to form an image of the pattern of the micromirror array 35, asrepresented by the individual beams 41 (and the dark areas between thesebeams), on the active surface 15 of the substrate 12. The outgoing beams41 are directed along a main optical axis of the image former 11 thatextends between the micromirror device and the substrate. The substrate12 in the configuration shown in FIG. 1 is transparent, e.g., formed offused silica or soda lime glass or quartz, so that the light projectedthereon, illustratively represented by the lines labeled 49, passesthrough the substrate 12 without substantial attenuation or diffusion.

A preferred micromirror array 35 is the Digital Micromirror Device (DMD)available commercially from Texas Instruments, Inc. These devices havearrays of micromirrors (each of which is substantially a square withedges of 10 to 20 μm in length) which are capable of forming patternedbeams of light by electronically addressing the micromirrors in thearrays. Such DMD devices are typically used for video projection and areavailable in various array sizes, e.g., 640×800 micromirror elements(512,000 pixels), 640×480 (VGA; 307,200 pixels), 800×600 (SVGA; 480,000pixels); and 1024×768 (786,432 pixels). Such arrays are discussed in thefollowing article and patents: Larry J. Hornbeck, “Digital LightProcessing and MEMs: Reflecting the Digital Display Needs of theNetworked Society,” SPIE/EOS European Symposium on Lasers, Optics, andVision for Productivity and Manufacturing I, Besancon, France, Jun.10-14, 1996; and U.S. Pat. Nos. 5,096,279, 5,535,047, 5,583,688 and5,600,383. The micromirrors 36 of such devices are capable of reflectingthe light of normal usable wavelengths, including ultraviolet and nearultraviolet light, in an efficient manner without damage to the mirrorsthemselves.

The window of the enclosure for the micromirror array preferably hasanti-reflective coatings thereon optimized for the wavelengths of lightbeing used. Utilizing commercially available 600×800 arrays ofmicromirrors, encoding 480,000 pixels, with typical micromirror devicedimensions of 16 microns per mirror side and a pitch in the array of 17microns, provides total micromirror array dimensions of 13,600 micronsby 10,200 microns. By using a reduction factor of 5 through the opticssystem 44, a typical and readily achievable value for a lithographiclens, the dimensions of the image projected onto the substrate 12 arethus about 2,220 microns by 2040 microns, with a resolution of about 2microns. Larger images can be exposed on the substrate 12 by utilizingmultiple side-by-side exposures (by either stepping the flow cell 18 orthe image projector 11), or by using a larger micromirror array. It isalso possible to do one-to-one imaging without reduction as well asenlargement of the image on the substrate, if desired.

The projection optics 44 may be of standard design, since the images tobe formed are relatively large and well away from the diffraction limit.The lenses 45 and 46 focus the light in the beam 41 passed through theadjustable iris 47 onto the active surface of the substrate. Theprojection optics 44 and the beam splitter 32 are arranged so that thelight deflected by the micromirror array away from the main optical axis(the central axis of the projection optics 44 to which the beams 41 areparallel), illustrated by the beams labeled 40 (e.g., 10° off axis) falloutside the entrance pupil of the projection optics 44 (typically0.5/5=0.1; 10° corresponds to an aperture of 0.17, substantially greaterthan 0.1). The iris 47 is used to control the effective numericalaperture and to ensure that unwanted light (particularly the off-axisbeams 40) are not transmitted to the substrate. Resolution of dimensionsas small as 0.5 microns are obtainable with such optics systems. Formanufacturing applications, it is preferred that the micromirror array35 be located at the object focal plane of a lithographic I-line lensoptimized for 365 nm. Such lenses typically operate with a numericalaperture (NA) of 0.4 to 0.5, and have a large field capability

The micromirror array device 35 may be formed with a single line ofmicromirrors (e.g., with 2,000 mirror elements in one line) which isstepped in a scanning system. In this manner the height of the image isfixed by the length of the line of the micromirror array but the widthof the image that may be projected onto the substrate 12 is essentiallyunlimited. By moving the stage 18 which carries the substrate 12, themirrors can be cycled at each indexed position of the substrate todefine the image pattern at each new line that is imaged onto thesubstrate active surface.

Various approaches may be utilized in the fabrication of the DNA probes16 on the substrate 12, and are adaptations of microlithographictechniques. In a “direct photofabrication approach,” the glass substrate12 is coated with a layer of a chemical capable of binding thenucleotide bases. Light is applied by the projection system 11,deprotecting the OH groups on the substrate and making them availablefor binding to the bases. After development, the appropriate nucleotidebase is flowed onto the active surface of the substrate and binds to theselected sites using normal phosphoramidite DNA synthesis chemistry. Theprocess is then repeated, binding another base to a different set oflocations. The process is simple, and if a combinatorial approach isused the number of permutations increases exponentially. The resolutionlimit is presented by the linear response of the deprotection mechanism.Because of the limitations in resolution achievable with this method,methods based on photoresist technology may be used instead, asdescribed, e.g., in McGall, et al., supra. In the indirectphotofabrication approach, compatible chemistries exist with a two-layerresist system, where a first layer of, e.g., polyimide acts as aprotection for the underlying chemistry, while the top imaging resist isan epoxy-based system. The imaging step is common to both processes,with the main requirement being that the wavelength of light used in theimaging process be long enough not to excite transitions (chemicalchanges) in the nucleotide bases (which are particularly sensitive at280 nm). Hence, wavelengths longer than 300 nm should be used. 365 nm isthe I-line of mercury, which is the one used most commonly in waferlithography.

Another form of the array synthesizer apparatus 10 is shown in asimplified schematic view in FIG. 2. In this arrangement, thebeamsplitter 32 is not used, and the light source 25, optional filter26, and condenser lens 28 are mounted at an angle to the main opticalaxis (e.g., at 20° to the axis) to project the beam of light 30 onto thearray of micromirrors 36 at an angle. The micromirrors 36 are orientedto reflect the light 30 into off axis beams 40 in a first position ofthe mirrors and into beams 41 along the main axis in a second positionof each mirror. In other respects, the array synthesizer of FIG. 2 isthe same as that of FIG. 1.

A more detailed view of a preferred array synthesizer apparatus whichuses the off-axis projection arrangement of FIG. 2 is shown in FIG. 3.In the apparatus of FIG. 3, the source 25 (e.g., 1,000 W Hg arc lamp,Oriel 6287, 66021), provided with power from a power supply 50 (e.g.,Oriel 68820), is used as the light source which contains the desiredultraviolet wavelengths. The filter system 26 is composed, for example,of a dichroic mirror (e.g., Oriel 66226) that is used to absorb infraredlight and to selectively reflect light of wavelengths ranging from 280to 400 nm. A water-cooled liquid filter (e.g., Oriel 6127) filled withdeionized water is used to absorb any remaining infrared. A coloredglass filter (Oriel 59810) or an interference filter (Oriel 56531) isused to select the 365 nm line of the Hg lamp 25 with a 50% bandwidth ofeither 50 nm or 10 nm, respectively. An F/1 two element fused silicacondenser (Oriel 66024) is used as the condenser 28, and with twoplano-convex lenses 52 (Melles Griot 01LQP033 and Melles Griot01LQP023), forms a Kohler illumination system. This illumination systemproduces a roughly collimated uniform beam 30 of 365 nm light with adiameter just large enough to encompass the 16 mm×12 mm active area ofthe micromirror array device 35. This beam 30 is incident onto thedevice 35 at an angle of 20° measured from the normal to the face of thedevice. The micromirror array device 35 is located approximately 700 mmaway from the last filter. When the micromirrors are in a firstposition, the light in the beam 30 is deflected downwardly and out ofthe system. For example, in this micromirror device the mirrors in theirfirst position may be at an angle of −10° with respect to the normal tothe plane of the micromirrors to reflect the light well away from theoptical axis. When a micromirror is controlled to be deflected in asecond position, e.g., at an angle of +10° with respect to the normal tothe plane of the micromirrors, the light reflected from suchmicromirrors in the second position emerges perpendicularly to the planeof the micromirror array in the beam 41. The pattern formed by the lightreflected from the micromirrors in their second position is then imagedonto the active surface 15 of a glass substrate 12 enclosed in a flowcell 18 using a telecentric imaging system composed of two doubletlenses 45 and 46 and an adjustable aperture 47. Each of the doubletlenses 45 and 46 is composed of a pair of plano-convex lenses (e.g.,Melles Griot 01LQP033 and 01LQP037) put together with the curvedsurfaces nearly touching. The first doublet lens is oriented so that theshorter focal length (01LQP033) side is towards the micromirror arraydevice 35, and the second doublet is oriented so that its longer focallength (01LQP037) side is toward the micromirror array device 35.Doublets composed of identical lenses may be used, in which case eitherside may face the micromirror array device. The adjustable aperture 47,also called a telecentric aperture, is located at the back focal planeof the first doublet. It is used to vary the angular acceptance of theoptical system. Smaller aperture diameters correspond to improvecontrast and resolution but with correspondingly decreased intensity inthe image. As illustrated in FIG. 3, a standard DNA synthesizer 55supplied with the requisite chemicals can be connected by the tubes 20and 21 to the flow cell 18 to provide the desired sequence of chemicals,either under independent control or under control of the computer 38. Atypical diameter for the aperture 47 is about 30 nm. An illustrative raydiagram showing the paths of light through the lenses 45 and 46 is shownin FIG. 4 for this type of refractive optical system. Fans of raysoriginating at the center of the object (the micromirror device face),at the edge, and at an intermediate location are shown. The opticalsystem forms an inverted image of the face of the micromirror arraydevice.

Another embodiment of the array synthesizer apparatus using reflectiveoptics is shown in FIG. 5. An exemplary system utilizes a 1,000 W Hg arclamp 25 as a light source (e.g., Oriel 6287, 66021), with a filtersystem formed of a dichroic mirror (e.g., Oriel 66228) that absorbsinfrared light and selectively reflects light of wavelengths rangingfrom 350 to 450 nm. An F/1 two element fused silica condenser lens(Oriel 66024) is used to produce a roughly collimated beam of light 30containing the 365 nm line but excluding undesirable wavelengths aroundand below 300 nm. A Kohler illumination system may optionally also beused in the apparatus of FIG. 5 to increase uniformity and intensity.The beam 30 is incident onto the micromirror array device 35 which hasan active area of micromirrors of about 16 mm×12 mm and which is locatedabout 210 nm from the snout of the UV source 25, with the beam 30striking the planar face of the micromirror device 35 at an angle of 20°with respect to a normal to the plane of the array. The light reflectedfrom the micromirrors in a first position of the micromirrors, e.g.,−10° with respect to the plane of the array, is directed out of thesystem, whereas light from micromirrors that are in a second position,e.g., +10° with respect to the plane of the array, is directed in thebeam 41 toward a reflective telecentric imaging system composed of aconcave mirror 60 and a convex mirror 61. Both mirrors are preferablyspherical and have enhanced UV coating for high reflectivity. Afterexecuting reflections from the mirrors 60 and 61, the beam 41 may beincident upon another planar mirror 63 which deflects the beam towardthe flow cell 18. The light reflected from the micromirrors is imagedonto the active surface of a glass substrate enclosed in the flow cell18. A telecentric aperture (not shown in FIG. 5) may be placed in frontof the convex mirror. The beam 41 first strikes the concave mirror, thenthe convex mirror, and then the concave mirror again, with the planarmirror 63 optionally being used to deflect the light 90° to direct it tothe flow cell 18. For the system shown, the concave mirror 60 may have adiameter of 152.4 mm, and a spherical mirror surface radius of 304.8 mm(ES F43561), and the convex mirror may have a diameter of 25 mm, and aspherical mirror surface radius of 152.94 mm (ES F45625). Ideally, theradius of curvature of the concave mirror is twice that of the convexmirror. Such reflective optical systems are well known andconventionally used in optical lithography in “MicroAlign” type systems.See, e.g., A. Offner, “New Concepts in Projection Mask Aligners,”Optical Engineering, Vol. 14, pp. 130-132 (1975), and R. T. Kerth, etal., “Excimer Laser Projection Lithography on a Full-Field ScanningProjection System,” IEEE Electron Device Letters, Vol. EDL-7(5), pp.299-301 (1986).

FIG. 6 illustrates image formation for the optical system of FIG. 5.Fans of rays originating in the center of the object (the micromirrorarray device), at the edge, and at an intermediate position are shown inFIG. 6. The rays reflect first from the concave mirror 60, then from theconvex mirror 61, then from the concave mirror 60 again, to form aninverted image of the face of the micromirror array device. The planarmirror 63 is not included in the diagram of FIG. 6. A telecentricaperture (not shown) may be placed in front of the convex mirror.

The refractive or reflective optical systems are both preferably“symmetric” to minimize aberrations such as coma and sphericalaberration via cancellation. The foregoing reflective system has ahigher numerical aperture which yields higher intensity. Both of thetelecentric optical systems of FIGS. 3 and 5 are 1:1 imaging systems. Areflective system has the potential advantages of eliminating chromaticaberration and providing higher resolution, as well as being compact andless expensive. A preferred system for doing 1:1 imaging would be aWynne-Dyson type system which combines concave mirror with lenses andprisms. It has a very high numerical aperture which enhances intensity.See, e.g., F. N. Goodall, et al., “Excimer Laser Photolithography with1:1 Wynne-Dyson Optics,” SPIE Vol. 922, Optical/Laser Microlithography,1988; and B. Ruff, et al., “Broadband Deep-UV High NA PhotolithographySystem,” SPIE Vol. 1088, Optical/Laser Microlithography II (1989).

More detailed views of a reaction chamber flow cell 18 that may beutilized with the apparatus of the invention is shown in FIGS. 7 and 8.The exemplary flow cell 18 in these figures includes an aluminum housing70, held together by bolts 71, having an inlet 73 connected to an inputport line 20 and an outlet 75 converted to an output port line 21. Asillustrated in the cross-sectional view of FIG. 8, the housing 70includes a lower base 78 and an upper cover section 79 which are securedtogether over the substrate with the bolts 71. The substrate 12, e.g., atransparent glass slide, is held between the upper plate 79 and acylindrical gasket 81 (e.g., formed of Kal Rezθ), which in turn issupported on a nonreactive base block 82 (e.g., Teflonθ), with an inletchannel 85 extending from the inlet 73 to a sealed reaction chamber 88formed between the substrate 12 and the base block 82 that is sealed bythe gasket, and with an outlet channel 89 extending from the reactionchamber 88 to the outlet 75. The bolts 71 can be screwed and unscrewedto detachably secure the substrate 12 between the cover section and thebase to allow the substrate to be replaced with minimal displacement ofthe base of the flow cell. Preferably, as shown in FIG. 8, a rubbergasket 90 is mounted at the bottom of the plate 79 to engage against thesubstrate at a peripheral region to apply pressure to the substrateagainst the gasket 81. If desired, the flow cell may also be used as ahybridization chamber during readout.

An exemplary process for forming DNA probes is illustrated with respectto the schematic diagrams of FIGS. 9-14. FIG. 9 illustrates the coatingof the substrate 12, having a silane layer 95 forming the active surface15 thereof, with the photolabile linker molecule MENPOC-HEG coated onthe silane layer using standard phosphoramidite chemistry.MENPOC-HEG-CEP=18-O-[(R,S)-(1-(3,4-(Methylenedioxy)-6-nitrophenyl)ethoxy)carbonyl]-3,6,9,12,15,18-hexaoxaoctadec-1-ylO′-2-cyanoethyl-N,N-Diisopropylphosphoramidite. The silane layer wasmade from N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide. At the stepshown in FIG. 9, the substrate can be exposed to light and active freeOH groups will be exposed in areas that have been exposed to light.

FIG. 10 illustrates the photo-deprotection of the MENPOC-HEG linker andthe production of free OH groups in the area 100 that is exposed tolight. FIG. 11 illustrates the coupling of FluorePrimeθ fluoresceinamidite to free OH groups produced from photo-deprotection ofMENPOC-HEG. FIG. 12 illustrates the coupling of DMT-nucleotide to freeOH groups produced from photo-deprotection of MENPOC-HEG linker. FIG. 13illustrates the step of acid deprotection of DMT-nucleotides in the area100 exposed to light. FIG. 14 illustrates the hybridization of poly-Aprobe labeled with fluorescein with poly-T oligonucleotides synthesizedfrom DMT-nucleotide-CEPs.

It is understood that the invention is not confined to the particularembodiments set forth herein as illustrative, but embraces all suchmodified forms thereof as come within the scope of the following claims.

1. A method of projecting a pattern onto a surface comprising the stepsof: (a) providing a substrate with an active surface to which protectedlinker molecules have been applied; (b) projecting a two-dimensionallight image onto the active surface of the substrate using projectionoptics comprising reflective optical elements to illuminate pixel siteson the active surface to photodeprotect the linker molecules thereon,wherein the reflective optical elements provide telecentric projectionoptics and wherein the step of projecting the two-dimensional image ontothe active surface of the substrate comprises: (i) providing amicromirror device comprising a two-dimensional array of electronicallyaddressable micromirrors, each of which can be selectively tiltedbetween one of at least two separate positions, and providing signals tothe micromirror device to select a pattern of the micromirrors in thetwo-dimensional array which are to reflect light onto the activesurface; and (ii) projecting light from a light source onto themicromirror array and reflecting the light from the mirrors of themicromirror array onto the projection optics to project thetwo-dimensional image onto the active surface, and further wherein thestep of projecting the two-dimensional light image does not use alithographic mask.
 2. The method of claim 1, wherein the reflectiveoptical elements comprise a concave mirror and a convex mirror, theconcave mirror reflecting light to the convex mirror which reflects thelight back to the concave mirror which reflects the light to the activesurface where it is imaged.
 3. The method of claim 2, wherein thereflective optical elements further comprise a planar mirror whichreflects the light from the concave mirror to the active surface.
 4. Themethod of claim 2, wherein the projection optics further comprise atelecentric aperture placed in front of the convex mirror.
 5. The methodof claim 2, wherein the radius of curvature of the concave mirror istwice that of the convex mirror.
 6. A method of synthesizing arrays ofDNA probes comprising carrying out the method of projecting a patternonto a surface according to claim 2, the method further comprising: (c)providing a fluid containing an appropriate nucleotide base to theactive surface of the substrate and binding the nucleotide base to thephotodeprotected linker molecules.
 7. The method of claim 1, wherein thesubstrate is transparent and the light image is projected through asurface of the substrate that is opposite to the active surface.
 8. Themethod of claim 1, further comprising collimating the light from thelight source to provide a collimated beam projected onto the micromirrorarray at an oblique angle to a main optical axis that extends from themicromirror array to the substrate, and wherein in one position of eachmicromirror the light is reflected along the main optical axis onto theprojection optics and in a second position of each micromirror the lightfrom the source is reflected at an angle off the main optical axis andaway from the substrate.
 9. The method of claim 1, further comprisingfiltering the light from the light source, whereby only desiredwavelengths of light are passed through to the micromirror array. 10.The method of claim 9, wherein the light from the light source comprisesultraviolet or near-ultraviolet light and the desired wavelengths thatare passed are in the range of ultraviolet or near-ultravioletwavelengths.
 11. The method of claim 9, wherein the light from the lightsource is filtered by a filter comprising a dichroic mirror thatreflects the desired wavelengths and passes wavelengths to be blocked.12. The method of claim 1, further comprising a computer connected tothe micromirror device to provide command signals to control thedeflection of the mirrors in the micromirror array to provide a desiredpattern for projection onto the active surface.
 13. The method of claim1, wherein the image that is patterned onto the active surface of thesubstrate is reduced in size with respect to the size of the array ofmicromirrors.
 14. A method of synthesizing arrays of DNA probescomprising carrying out the method of projecting a pattern onto asurface according to claim 1, the method further comprising: (c)providing a fluid containing an appropriate nucleotide base to theactive surface of the substrate and binding the nucleotide base to thephotodeprotected linker molecules.
 15. The method of claim 14, furthercomprising: (d) projecting a new two-dimensional light image onto theactive surface of the substrate to illuminate pixel sites on the activesurface to photodeprotect linker molecules or bound nucleotide bases;and (e) providing a fluid containing an appropriate nucleotide base tothe active surface of the substrate and binding the nucleotide base tothe photodeprotected linker molecules or bound nucleotide bases.
 16. Themethod of claim 15, wherein steps (d) and (e) are repeated a selectednumber of times to build up a selected number of levels of nucleotidebases in a DNA probe array on the substrate.
 17. The method of claim 14,wherein the nucleotide base is flowed over the active surface in step(c) and bound to the linker molecules utilizing phosphoramidate DNAsynthesis.
 18. The method of claim 14, wherein the active surface of thesubstrate is enclosed in a flow cell comprising a sealed reactionchamber, an input port and an output port.
 19. The method of claim 18,wherein the flow cell comprises a housing comprising a lower base, anupper cover section and a gasket mounted on the base, wherein thesubstrate is a transparent glass slide secured between the upper coversection and the lower base to define the sealed reaction chamber betweenthe substrate and the lower base that is sealed by the gasket, andchannels extending through the housing from the input port to thereaction chamber and from the reaction chamber to the output port, theactive surface of the substrate facing the sealed reaction chamber.